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Applied Microbiology and Biotechnology

, Volume 90, Issue 6, pp 1923–1932 | Cite as

High-yield production of hydrophobins RodA and RodB from Aspergillus fumigatus in Pichia pastoris

  • Mona Højgaard PedersenEmail author
  • Irina Borodina
  • Jacob Lange Moresco
  • Winnie Edith Svendsen
  • Jens Christian Frisvad
  • Ib Søndergaard
Biotechnological Products and Process Engineering

Abstract

Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins in Pichia pastoris and present fed-batch fermentation yields of 200–300 mg/l fermentation broth. Protein bands of expected sizes were detected by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy and fed-batch production using P. pastoris may be transferred to hydrophobins from other species.

Keywords

Hydrophobin RodA RodB Aspergillus fumigatus Pichia pastoris Protein expression Fed-batch fermentation 

Notes

Acknowledgements

This study was funded by The Danish Research Council for Technology and Production. We also gratefully acknowledge support to this project from The Danish Council for Independent Research, Technology and Production Sciences, Grant No. 09-064967. We furthermore thank Anne Blicher at Center for Enzyme and Protein Chemistry, DTU Systems Biology, Technical University of Denmark for making the amino acid analysis.

Conflicts of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Mona Højgaard Pedersen
    • 1
    Email author
  • Irina Borodina
    • 1
  • Jacob Lange Moresco
    • 2
  • Winnie Edith Svendsen
    • 2
  • Jens Christian Frisvad
    • 1
  • Ib Søndergaard
    • 1
  1. 1.Center for Microbial BiotechnologyTechnical University of Denmark, DTU Systems BiologyKgs. LyngbyDenmark
  2. 2.DTU NanotechTechnical University of DenmarkKgs. LyngbyDenmark

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